MeDIP followed by Real Time - (Dec/19/2008 )

I am new to this forum and have a new project on epigenetics. Here, i am trying to start with DNA methylation study on some specific genes by MeDIP (CHIP using antibody against 5-MeC) followed by Real Time PCR with TaqMan using relative standard curve.
my questions are,

if i just want to compare mathylation level of one gene among 10 samples by the value i will get doing Ct of IP vs Ct of input for each sample, why do i need to use positive control (i can understand + control is to know whether expt is working properly ) Is there any other reason?

I use skin fibroblast. In making relative standard curve, do i need to use serial dilutions of genomic DNA from different cells ? and do i need to make std curve for all of my target genes and + control?

In my case, it might be possible, the CpG island in the promoter region i am targetting to make my primers, the methylation level is high in other part of the same CpG island, Am i right? in that case i guss, i need to generate primers for that part also, Is it? Correct me if i am wrong?

looking forward to know the answers,
thanks.

hi edu,

if you are doing a medip you can always compare Ct's relative to the input fraction, the starting DNA before you perform the IP. as opposed to a standard curve, this way, you control for potential CNV as well.

Nick

Thanks Nick for your ans. I have another query. I found here, somebody mentioned bisulfite sequencing is more specific than Medip. I know, sequencing is good, but it will be huge work if you want to analyse 15 genes in 20 samples. and also you can never be sure that conversion worked 100%,
but why MeDIP is less specific , i cant understand, I am going to use monoclonal antobody. so this one also binds to something nonspecific/ non methylated part or something else?

and is it a good idea to use Kit for MeDIP or just follow the traditional protocol?
thanks.

MeDip is a rather blunt instrument to look at methylation and there are papers that say that there is a CpG density dependence for proper binding to the antibody, meaning there will be biases to more dense regions of the genome.

Furthermore, you need to ensure that your DNA is denatured as the antibody only binds to single stranded moeities.

Bisulfite sequencing is another approach which I feel is complementary to MeDIP and will confirm the MeDIP enrichment findings.

N